The central goal of this project is to define the mechanisms by which keratinocytes and sensory neurons communicate in vivo under naive conditions and after tissue injury, to convey mechanical information to the CNS. It is well known that sensory afferent fibers respond to external mechanical stimuli and convey this information to spinal cord circuits that project to brain regions. The peripheral terminals of cutaneous sensory neurons are largely assumed to be the sole site of transduction for evoked mechanical stimuli. However, cutaneous mechanical stimuli first impact keratinocytes which comprise 95% of the epidermis. Anatomically, keratinocytes are closely apposed to sensory nerve terminals and appear to make synapse-like contacts with them. Although isolated keratinocytes respond to mechanical stimuli, the in vivo roles of keratinocytes in detecting and conveying baseline mechanotransduction have not been investigated and are important for understanding normal cutaneous mechanotransduction. In the opposite direction, after tissue injury, peripheral sensory nerve terminals release neurotransmitters and neuropeptides that induce neurogenic inflammation by acting on multiple cell types including blood vessels and immune cells. Whether these neuronally-released molecules act on receptors on keratinocytes to contribute to the mechanical sensitization of primary afferent terminals in vivo during tissue injury has not been mechanistically investigated and is important because this process may contribute to sensitization during skin inflammation or disease. Our preliminary findings suggest that in non-injured skin, mechanical stimulation evokes local ATP release, and hydrolysis of cutaneous ATP elevates mechanical thresholds. Further, mechanically-evoked ATP release is increased after tissue injury, and P2X4 receptors are involved in the mechanical coding. From these data, we hypothesize that keratinocytes and cutaneous sensory neurons communicate with each other in a bidirectional manner. This proposal will explore the mechanisms of bidirectional signaling through two Specific Aims: Aim 1 will test the hypothesis that the mechanical sensitivity of nave skin depends on ATP release from keratinocytes which signals through P2X4 receptors on sensory neurons. Aim 2 will examine the hypothesis that the mechanical sensitization after tissue injury involves sensory neuron release of factors that act on keratinocyte NMDA or NK1 receptors, and amplify keratinocyte ATP release. These studies will illuminate basic touch transduction mechanisms, lay a foundation for understanding dysfunctional signaling processes during cutaneous inflammatory pain and disease, and potentially reveal targets for topical therapeutics that are easily applied and may limit CNS side effects.